Meeting
attendants: Jakob, Julia, Manuel, Rüdiger, Sandra, Theo, Tobi
time: 9:30 - 11:00
green light receptor
already done:
- CcaS: first quick change PCR removed the SpeI restriction site, EcoRI didn't work
- CcaR: first quick change PCR didn't work (EcoRI restriction site not removed), wrong PCR program
To-do:
- do the second quick change with the changed Ccas to remove the EcoRI restriction site
- repeat the quick change with the CcaR
- after the quick changes do a Gibson assembly with pcyA and ho1
- in parallel do 3A-assemblies to create the same construct as a backup plan
blue light receptor
already done:
- ordered new primers (the third pair)
- first primer pair had the overhang, second without overhang, third primer pair now without overhang and position shifted
To-do:
- try the gibson assembly again with the third primer pair
red light receptor
already done:
- 3A of pcyA and terminator sent for sequencing
- cph8 was sent for sequencing
- julia transformed the ompR promotor
To-do:
- add a Promotor-RBS to the pcyA-Terminator
- if the sequence is OK, amplify the part with the overhangs
- add terminator and Promotor-RBS
- do a miniprep of the ompR promotor
Lysis cassette
already done:
- 3A-assembly of the lysis casette
- 3A-assembly oth the GFP-PBD
- quick change PCR of the lysis casette (11 bases to insert the RBS)
To-do:
- do a miniprep from all three attempts
Precipitator
already done:
To-do:
other stuff
- start creating nice pictures to explain your subproject and write text to explain the pictures
- Sandra organized Freehand from the MPI, it is installed on both mac books
- give-aways: digital postcard, real postcard, fluorescent protein
- Tobi will ask at the financial department about the croud funding money
- Rüdiger will get us more media attention
- cooperation with other german teams: ask munich to do an appointment for a skype conforence about the light inducible promotors
- cooperation with Upsala: find an appointment for the skype talk
green light receptor
Quickchange
Investigators: Jakob
PCR
| Name:
Jakob
| Date:
05.08.2011
|
| Continue from Experiment: 05.08.2011
Quickchange of Spe1 (Sequencing was correct)
|
| Project Name:
Green light receptor
|
PCR-Mixture for one Reaction:
For a 50 µl reaction use
| 32,5µl
| H20
| Name
|
| 10µl
| 5x Phusion Buffer
| of Primer
|
| 2.5µl
| Primer fw
| P14 (1:10)
|
| 2.5µl
| Primer dw
| P15 (1:10)
|
| 1µl
| dNTPs
| of Template DNA
|
| 1µl
| DNA-Template
| CcaS (120ng/µl) and CcaS (130ng/µl) dilute to 5ng/µl
|
| 0.5 µl
| Phusion (add in the end)
|
|
What program do you use?
| 95 °C – 5’
| 95°C – 15 ‘’
| X 20
| 72°C – 5’
| 4°C – ∞
|
| 55°C – 15 ‘’
|
| 72°C – 1,15’ + 2’’/Cycle
|
To confirm the PCR-Product has the correct size, load 2 µl of the sample onto an agarose-gel.
How did you label the PCR-Product, where is it stored and what do you do next?
Labeled as Jakob1 and Jakob2 in the fridge
Digest quickchange PCR with Dpn1
Investigator: Jakob
5µl Buffer NEB4
5µl BSA (10x)
2µl Dpn1
38µl DNA from quickchange (CcaR)
incubate at 37°C for 1,5 h
inactivate at 80°C for 20 min.
blue light receptor
Gradient PCR of Lovtap with new primer
Investigators: Sandra
Primer:
- P38 (former name:P36) LOVdw
- P42 LOVtap up
DNA template:
PCR program:
- "Lovtap ohne Überhänge" with temperature gradient during the annealing step from 50°C-60°C. The elongation step and the final elongation step were extended.
File:Freiburg 2011.jpg
red light receptor
NAME OF YOUR EXPERIMENT
Investigators:NAME
Lysis cassette
NAME OF YOUR EXPERIMENT
Investigators:NAME
Precipitator
NAME OF YOUR EXPERIMENT
Investigators: NAME
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